Project Summary/Abstract During maximal exercise or ischemia, blood supply is insufficient to meet the metabolic needs of skeletal muscle, and so metabolic by-products are produced and accumulate. Sensory nerves (afferents) that innervate muscle respond to these changes in the metabolic milieu, and send signals centrally. These signals can initiate exercise-mediated reflexes, and sometimes they signal muscle pain. Multiple chemicals have been shown to activate muscle afferents, but the molecular identity of the receptors that sense these chemicals are not well understood. Acid-Sensing Ion Channels (ASICs) are suited to sense extracellular chemical changes in muscle. Our overall goal is to understand the role of ASICs as receptors in muscle afferents during the related conditions of muscle ischemia and exercise. A strength of our proposal is that we have developed tools to unravel the components of muscle afferent activation at the molecular level. These studies will advance our understanding of how muscle afferents sense pain and exercise, and provide a target for future studies and potential therapies. Specifically, our Aims are to: 1) determine the subunit composition of ASIC channels in skeletal muscle afferents; 2) define the mechanisms by which ASICs are modulated by other metabolites; and 3) determine the role of ASICs metaboreceptors during ischemia and exercise. These studies will provide significant insight into the molecular mechanisms that underlie painful conditions such as claudication from peripheral vascular disease, pain associated with sickle cell disease, fibromyalgia, muscle injury, and other painful muscle conditions. Additionally, our results will provide molecular insight into the mechanisms how muscle afferents sense metabolic changes in muscle and activate exercise-mediated reflexes. Moreover, our studies have implications for common cardiovascular diseases. Heart failure is now the most common cause for hospitalization in the United States. It is well understood, that increases in the sympathetic nervous system plays a major role in the progression of the disease, and much of our pharmacological treatments are aimed at blocking this sympathoexcitation. Muscle afferent activity is one of the major mechanisms contributing to this high sympathetic tone, and alterations of muscle reflexes are believed to contribute to the poor exercise tolerance of patients with heart failure. Increasing evidence points to ASICs as a major sensor of the metabolic milieu of muscle, and better understanding of their role could provide a molecular target to treat muscle pain and cardiovascular diseases.